San taq pcr mix

Well-documented 4 primer pairs respectively for the 4 intergenic spacers (Table 2) were adopted. The spacers were PCR amplified in a 25μl reaction system consisted of 2×SanTaq PCR Mix (Sangon, Shanghai, China) 11μl, primers (10μmol/L) each 1μl, DNA template 1μl, ddH₂O 11μl, with the program of pre-denaturation at 94°C for 5min, denaturation at 94°C for 30s, annealing for 30s, extension at 72°C for 30s, 35 cycles, and a final extension at 72°C for 10min. Products were electrophoresed by agarose gel, purified and then bidirectionally sequenced by dideoxy chain termination (Sangon, Chengdu, China).

The RNAprep Pure Cell/Bacteria Kit (Tiangen Biotech, Beijing, China) was used to extract total RNA from intrahepatic immune cells according to the manufacturer’s specifications. Then, 200 ng of total RNA was reverse-transcribed into cDNA using a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Penzberg, Germany) on a C1000 Touch^TM Thermal Cycler (Bio-Rad Inc., Hercules, CA, United States). Two-round nested amplicon arm-PCR with specific primers was performed using 2 × SanTaq PCR Mix (Sangon Biotech, Shanghai, China) as previously described (Liang et al., 2018 (link)). Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, United States). Purified amplicons were paired-end sequenced (PE250) on the Illumina platform according to standard protocols.
The clean data were harvested by filtering the low-quality sequences. The V, D, and J segments of the TCRβ consensus sequences were identified using the BLAST software (v2.2.25) in the international ImMunoGeneTics (IMGT) information system (IMGT)¹ by a standard algorithm. The Gini index, Shannon diversity, and CDR3 clustering were calculated using the QIIME (version 1.9.1).